The invention relates to the discovery of various active forms of xcex2-secretase, an enzyme that cleaves xcex2-amyloid precursor protein (APP) at one of the two cleavage sites necessary to produce xcex2-amyloid peptide (Axcex2). The invention also relates to inhibitors of this enzyme, which are considered candidates for therapeutics in the treatment of amyloidogenic diseases such as Alzheimer""s disease. Further aspects of the present invention include screening methods, assays, and kits for discovering such therapeutic inhibitors, as well as diagnostic methods for determining whether an individual carries a mutant form of the enzyme.
Alzheimer""s disease is characterized by the presence of numerous amyloid plaques and neurofibrillatory tangles present in the brain, particularly in those regions of the brain involved in memory and cognition. xcex2-amyloid peptide (Axcex2) is a 39-43 amino acid peptide that is major component of amyloid plaques and is produced by cleavage of a large protein known as the amyloid precursor protein (APP) at a specific site(s) within the N-terminal region of the protein. Normal processing of APP involves cleavage of the protein at point 16-17 amino acids C-terminal to the N-terminus of the xcex2-AP region, releasing a secreted ectodomain, xcex1-sAPP, thus precluding production of xcex2-AP. Cleavage by xcex2-secretase enzyme of APP between Met671 and Asp672 and subsequent processing at the C-terminal end of APP produces Axcex2 peptide, which is highly implicated in the etiology of Alzheimer""s pathology (Seubert, et al., in Pharmacological Treatment of Alzheimer""s disease, Wiley-Liss, Inc., pp. 345-366, 1997; Zhao, J., et al. J. Biol. Chem. 271: 31407-31411, 1996).
It is not clear whether xcex2-secretase enzyme levels and/or activity is inherently higher than normal in Alzheimer""s patients; however, it is clear that its cleavage product, Axcex2 peptide, is abnormally concentrated in amyloid plaques present in their brains. Therefore, it would be desirable to isolate, purify and characterize the enzyme responsible for the pathogenic cleavage of APP in order to help answer this and other questions surrounding the etiology of the disease. In particular, it is also desirable to utilize the isolated enzyme, or active fragments thereof, in methods for screening candidate drugs for ability to inhibit the activity of xcex2-secretase. Drugs exhibiting inhibitory effects on xcex2-secretase activity are expected to be useful therapeutics in the treatment of Alzheimer""s disease and other amyloidogenic disorders characterized by deposition of Axcex2 peptide containing fibrils.
U.S. Pat. No. 5,744,346 (Chrysler, et al.) describes the initial isolation and partial purification of xcex2-secretase enzyme characterized by its size (apparent molecular weight in the range of 260 to 300 kilodaltons when measured by gel exclusion chromatography) and enzymatic activity (ability to cleave the 695-amino acid isotype of xcex2-amyloid precursor protein between amino acids 596 and 597). The present invention provides a significant improvement in the purity of xcex2-secretase enzyme, by providing a purified xcex2-secretase enzyme that is at least 200 fold purer than that previously described. Such a purified protein has utility in a number of applications, including crystallization for structure determination. The invention also provides methods for producing recombinant forms of xcex2-secretase enzymes that have the same size and enzymatic profiles as the naturally occurring forms. It is a further discovery of the present invention that human xcex2-secretase is a so-called xe2x80x9caspartylxe2x80x9d (or xe2x80x9casparticxe2x80x9d) protease.
This invention is directed to a xcex2-secretase protein that has now been purified to apparent homogeneity, and in particular to a purified protein characterized by a specific activity of at least about 0.2xc3x97105 and preferably at least 1.0xc3x97105 nM/h/xcexcg protein in a representative xcex2-secretase assay, the MBP-C125sw substrate assay. The resulting enzyme, which has a characteristic activity in cleaving the 695-amino acid isotype of xcex2-amyloid precursor protein (xcex2-APP) between amino acids 596 and 597 thereof, is at least 10,000-fold, preferably at least 20,000-fold and, more preferably in excess of 200,000-fold higher specific activity than an activity exhibited by a solubilized but unenriched membrane fraction from human 293 cells, such as have been earlier characterized.
In one embodiment, the purified enzyme is fewer than 450 amino acids in length, comprising a polypeptide having the amino acid sequence SEQ ID NO: 70 [63-452]. In preferred embodiments, the purified protein exists in a variety of xe2x80x9ctruncated formsxe2x80x9d relative to the proenzyme reed to herein as SEQ ID NO: 2 [1-501], such as forms having amino acid sequences SEQ ID NO: 70 [63-452], SEQ ID NO: 69 [63-501], SEQ ID NO: 67 [58-501], SEQ ID NO: 68 [58-452], SEQ ID NO: 58 [46-452], SEQ ID NO: 74 [22-452], SEQ NO: 58 [46-452]. More generally, it has been found that particularly useful forms of the enzyme, particularly with regard to the crystallization studies described herein, are characterized by an N-terminus at position 46 with respect to SEQ ID NO: 2 and a C-terminus between positions 452 and 470 with respect to SEQ ID NO: 2. and more particularly, by an N-terminus at position 22 with respect to SEQ ID NO: 2 and a C-terminus between positions 452 and 470 with respect to SEQ ID NO: 2. These forms are considered to be cleaved in the transmembrane xe2x80x9canchorxe2x80x9d domain. Other particularly useful purified forms of the enzyme include: SEQ ID NO: 43 [46-501], SEQ ID NO: 66 [22-501], and SEQ ID NO: 2 [1-501]. More generally, it is appreciated that useful forms of the enzyme have an N-terminal residue corresponding to a residue selected from the group consisting of residues 22, 46, 58 and 63 with respect to SEQ ID NO: 2 and a C-terminus selected from a residue between positions 452 and 501 with respect to SEQ ID NO: 2 or a C-terminus between residue positions 452 and 470 with respect to SEQ ID NO: 2. Also described herein are forms of enzyme isolated from a mouse, exemplified by SEQ ID NO: 65.
This invention is further directed to a crystalline protein composition formed from a purified xcex2-secretase protein, such as the various protein compositions described above. According to one embodiment, the purified protein is characterized by an ability to bind to the xcex2-secretase inhibitor substrate P10-P4xe2x80x2sta Dxe2x86x92V which is at least equal to an ability exhibited by a protein having the amino acid sequence SEQ ID NO: 70 [46-4191], when the proteins are tested for binding to said substrate under the same conditions. According to another embodiment, the purified protein forming the crystallization composition is characterized by a binding affinity for the xcex2-secretase inhibitor substrate SEQ ID NO: 72 (P10-P4xe2x80x2sta Dxe2x86x92V) which is at least 1/100 of an affinity exhibited by a protein having the amino acid sequence SEQ ID NO: 43 [46-501], when said proteins are tested for binding to said substrate under the same conditions. Proteins forming the crystalline composition may be glycosylated or deglycosylated.
The invention also includes a crystalline protein composition containing a xcex2-secretase substrate or inhibitor molecule, examples of which are provided herein, particularly exemplified by peptide-derived inhibitors such as SEQ ID NO: 78, SEQ ID NO: 72, SEQ ID NO: 81, and derivatives thereof. Generally useful inhibitors in this regard will have a Ki of no more than about 50 xcexcM to 0.5 mM.
Another aspect of the invention is directed to an isolated protein, comprising a polypeptide that (i) is fewer than about 450 amino acid residues in length, (ii) includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 75 [63-423] including conservative substitutions thereof, and (iii) exhibits xcex2-secretase activity, as evidenced by an ability to cleave a substrate selected from the group consisting of the 695 amino acid isotype of beta amyloid precursor protein (BAPP) between amino acids 596 and 597 thereof, MBP-C125 wt and MBP-C125sw. Peptides which fit these criteria include, but are not limited to polypeptides which include the sequence SEQ ID NO: 75 [63-423], such as SEQ ID NO: 58 [46-452], SEQ ID NO: 58 [46-452], SEQ ID NO: 58 [46-452], SEQ ID NO: 74[22-452], and may also include conservative substitutions within such sequences.
According to a further embodiment, the invention includes isolated protein compositions, such as those described above, in combination with a xcex2-secretase substrate or inhibitor molecule, such as MBP-C125wt, MBP-C125sw, APP, APPsw, and xcex2-secretase-cleavable fragments thereof. Additional xcex2-secretase-cleavable fragments useful in this regard are described in the specification hereof. Particularly useful inhibitors include peptides derived from or including SEQ ID NO: 78. SEQ ID NO: 81 and SEQ ID NO: 72. Generally, such inhibitors will have K1s of less than about 1 xcexcM. Such inhibitors may be labeled with a detectable reporter molecule. Such labeled molecules are particularly useful, for example, in ligand binding assays.
In accordance with a further aspect, the invention includes protein compositions, such as those described above, expressed by a heterologous cell. In accordance with a further embodiment, such cells may also co-express a xcex2-secretase substrate or inhibitor protein or peptide. One or both of the expressed molecules may be heterologous to the cell.
In a related embodiment, the invention includes antibodies that bind specifically to a xcex2-secretase protein comprising a polypeptide that includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 75 [63-423] including conservative substitutions thereof, but which lacks significant immunoreactivity with a protein a sequence selected from the group consisting of SEQ ID NO: 2 [1-501] and SEQ ID NO: 43 [46-5011].
In a further related embodiment, the invention includes isolated nucleic acids comprising a sequence of nucleotides that encodes a xcex2-secretase protein that is at least 95% identical to a protein selected from the group consisting of SEQ ID NO: 66 [22-501], SEQ ID NO: 43 [46-501], SEQ ID NO: 57 [1-419], SEQ ID NO: 74 [22-452], SEQ ID NO: 58 [46-452], SEQ ID NO: 59 [1-452], SEQ ID NO: 60 [1-420], SEQ ID NO: 67 [58-501], SEQ ID NO: 68 [58-452], SEQ ID NO: 69 [63-501], SEQ ID NO: 70 [63-452], SEQ ID NO: 75 [63-423], and SEQ ID NO: 71 [46-419], or a complementary sequence of any of such nucleotides. Specifically excluded from this nucleotide is a nucleic acid encoding a protein having the sequence SEQ ID NO: 2 [1-501].
Additionally, the invention includes an expression vector comprising such isolated nucleic acids operably linked to the nucleic acid with regulatory sequences effective for expression of the nucleic acid in a selected host cell, for heterologous expression. The host cells can be a eukaryotic cell, a bacterial cell, an insect cell or a yeast cell. Such cells can be used, for example, in a method of producing a recombinant xcex2-secretase enzyme, where the method further includes subjecting an extract or cultured medium from said cell to an affinity matrix, such as a matrix formed from a xcex2-secretase inhibitor molecule or antibody, as detailed herein.
The invention is also directed to a method of screening for compounds that inhibit Axcex2 production, comprising contacting a xcex2-secretase polypeptide, such as those full-length or truncated forms described above, with (i) a test compound and (ii) a xcex2-secretase substrate, and selecting the test compound as capable of inhibiting Axcex2 production if the xcex2-secretase polypeptide exhibits less xcex2-secretase activity in the presence of than in the absence of the test compound. Such an assay may be cell-based, with one or both of the enzyme and the substrate produced by the cell, such as the co-expression cell referred to above. Kits embodying such screening methods also form a part of the invention.
The screening method may further include administering a test compound to a mammalian subject having Alzheimer""s disease or Alzheimer""s disease like pathology, and selecting the compound as a therapeutic agent candidate if, following such administration, the subject maintains or improves cognitive ability or the subject shows reduced plaque burden. Preferably, such a subject is a comprising a transgene for human xcex2-amyloid precursor protein (xcex2-APP), such as a mouse bearing a transgene which encodes a human xcex2-APP, including a mutant variants thereof, as exemplified in the specification.
In a related embodiment, the invention includes secretase inhibitor compound selected according to the methods described above. Such compounds may be is selected, for example, from a phage display selection system (xe2x80x9clibraryxe2x80x9d), such as are known in the art. According to another aspect, such libraries may be xe2x80x9cbiasedxe2x80x9d for the sequence peptide SEQ ID NO: 97 [P10-P4xe2x80x2Dxe2x86x92V]. Other inhibitors include, or may be derived from peptide inhibitors herein identified, such as inhibitors SEQ ID NO: 78, SEQ ID NO: 72, SEQ ID NO: 78 and SEQ ID NO: 81.
Also forming part of the invention are knock-out mice, characterized by inactivation or deletion of an endogendous xcex2-secretase gene, such as genes encodes a protein having at least 90% sequence identity to the sequence SEQ ID NO: 65. The deletion or inactivation may be inducible, such as by insertion of a Cre-lox expression system into the mouse genome.
According to a further related aspect, the invention includes a method of screening for drugs effective in the treatment of Alzheimer""s disease or other cerebrovascular amyloidosis characterized by Axcex2 deposition. According to this aspect of the invention, a mammalian subject characterized by overexpression of xcex2-APP and/or deposition of Axcex2 is given a test compound selected for its ability to inhibit xcex2-secretase activity a xcex2-secretase protein according to claim 37. The compound is selected as a potential therapeutic drug compound, if it reduces the amount of Axcex2 deposition in said subject or if it maintains or improves cognitive ability in the subject. According to one preferred embodiment, the mammalian subject is a transgenic mouse bearing a transgene encoding a human xcex2-APP or a mutant thereof.
The invention also includes a method of treating a patient afflicted with or having a predilection for Alzheimer""s disease or other cerebrovascular amyloidosis. According to this aspect, the enzymatic hydrolysis of APP to Axcex2 is blocked by administering to the patient a pharmaceutically effective dose of a compound effective to inhibit one or more of the various forms of the enzyme described herein. According to another feature, the therapeutic compound is derived from a peptide selected from the group consisting of SEQ ID NO: 72, SEQ ID NO: 78, SEQ ID NO: 81 and SEQ ID NO: 97. Such derivation may be effected by the various phage selection systems described herein, in conjunction with the screening methods of the invention, or other such methods. Alternatively, or in addition, derivation may be achieved via rational chemistry approaches, including molecular modeling, known in the medicinal chemistry art. Such compounds will preferably be rather potent inhibitors of xcex2-secretase enzymatic activity, evidenced by a Ki of less than about 1-50 xcexcM in a MBP-C125sw assay. Such compounds also form the basis for therapeutic drug compositions in accordance with the present invention, which may also include a pharmaceutically effective excipient.
According to yet another related aspect, the invention includes a method of diagnosing the presence of or a predilection for Alzheimer""s disease in a patient. This method includes detecting the expression level of a gene comprising a nucleic acid encoding xcex2-secretase in a cell sample from said patient, and diagnosing the patient as having or having a predilection for Alzheimer""s disease, if said expression level is significantly greater than a pre-determined control expression level. Detectable nucleic acids, and primers useful in such detection, are described in detail herein. Such nucleic acids may exclude a nucleic acid encoding the preproenzyme [1-501]. The invention is further directed to method of diagnosing the presence of or a predilection for Alzheimer""s disease in a patient, comprising measuring xcex2-secretase enzymatic activity in a cell sample from said patient, and diagnosing the patient as having or having a predilection for Alzheimer""s disease, if said level enzymatic activity level is significantly greater than a predetermined control activity level. The diagnostic methods may be carried out in a whole cell assay and/or on a nucleic acid derived from a cell simple of said patient.
The invention also includes a method of purifying a xcex2-secretase protein enzyme molecule. According to this aspect, an impure sample containing xcex2-secretase enzyme activity with an affinity matrix which includes a xcex2-secretase inhibitor, such as the various inhibitor molecules described herein.